The principal investigator and his collaborators propose to investigate the importance of protein dynamics on the catalytic rate enhancement of enzymatic methyl transfer. While much attention in the field of enzymatic catalysis is given to transition state stabilization or to entropic arguments, evidence in support of the importance of protein dynamics to catalysis is scant. Proteins are certainly known to undergo various types of conformational changes; for example, evidence in support of correlated motions contributing to ligand binding is available. Similar correlated motions have been identified for several enzyme-catalyzed reactions using molecular dynamic simulations. The investigators propose to apply a combination of x-ray crystallography, MD simulations, and functional analyses to provide an experimental and theoretical basis for relating protein dynamics and enzyme reaction rates. They propose to investigate M.HhaI, a bacterial S-adenosyl methionine-dependent DNA cytosine C5 methyltransferase. S-adenosyl methionine-dependent enzymes are widespread in biology and modify nucleic acids, proteins, lipids, carbohydrates, as well as drugs. Mammalian and bacterial DNA methyltransferases are important drug targets for anticancer and antibiotic drugs, respectively. M.HhaI is the best understood S-adenosyl methionine-dependent methyltransferase of any type, since numerous high-resolution cocrystal structures are available and both the kinetic and chemical mechanisms are well known. The principal investigator and his collaborators identified structural elements that appear to be important for protein dynamics through inspection of both the M.HhaI-DNA cocrystal structure and MD analysis of the same structure. Several mutants have been prepared and are undergoing crystallographic, multiple conformer (MCA), and MD analyses. They propose to compare this structural and dynamic information with measurements of the methyl transfer rate. Data analysis will focus on correlations between methylation and 1) protein flexibility, 2) interatomic distances, 3) correlated motions, 4) frequency of near attack conformations (NACs), and 5) distribution if conformers. Multiple conformer analysis of the WT M.HhaI-DNA and mutant cocrystal structures will be used to design additional mutants to test the relationship between methylation and correlated motions. An extract-based screen will follow random mutagenesis of segments implicated in correlated motions; candidate mutants will be submitted to the structural and functional analyses. The combination of structural, molecular dynamic, and functional analyses should provide unique insights with potentially broad impact, into how correlated conformational changes contribute to catalysis. M.HhaI represents a class of enzymes with significant medical applications, and a better mechanistic property.